Cancer is a leading cause of death in humans and a major medical need is to be able to detect early signs of the disease and monitor the success of treatments. Serum tumor markers have been used for these purposes for more than 30 years. Thymidine kinase 1 protein (TK1) is a cytosolic enzyme involved in the synthesis of DNA precursors and it is only expressed in proliferating cells, i.e., in cells in the S phase of the cell cycle. This enzyme is a classic marker for cell proliferation and its regulation and properties have been studied extensively during the last 40 years (1). It was discovered that a form of TK1 was found in the serum of animals and humans with a high proportion of proliferating cells, e.g., in patients with some type of infection or tumor disease. Sensitive radio-isotope methods were developed and used to determine the level of active TK1 in serum from patients with different types of diseases (2-4). The clinical value of determination of serum TK1 as a tumor marker in monitoring hematologic cancers has been well established and a commercial cancer marker kit has been available (Prolifigen® TK-REA from DiaSorin) with applications in non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myeloic leukemia and multiple myeloma (2-4). Furthermore, measuring TK activity in cytosolic breast tumor extracts has given relatively good prognostic information as a tumor marker (5, 6). However, drawbacks of the Prolifigen® assay are that it is relatively complex and dependent on radio-isotopes and as a serum marker it is limited in use as only hematologic malignancies can be monitored.
More recently, an alternative non-radioactive TK activity assay was developed based on the high capacity of TK1 to phosphorylate the stable substrate analog 3′-azido-thymidine. The product of this reaction, AZTMP, was determined using polyclonal antibodies directed against AZTMP (7). A fully automated competitive chemiluminescence assay has been developed (Liason thymidine kinase) and is commercially available from DiaSorin AB for the same applications as those described above (8). A complex but sensitive non radioactive immunoassay (DiviTum®), based on the phosphorylation and incorporation of 5-bromo-deoxyuridine into an oligonucleotide, has recently been placed on the market for the follow up of lymphomas and leukemia by Biovica AB (9). The main drawbacks of these methods are the limitation of their application to hematologic malignancies and their dependence on time-consuming enzyme reaction measurements. Further, the Liason thymidine kinase assay requires the use of special instrumentation and the DiviTum® assay requires a long process time.
In recent years, several different monoclonal and polyclonal antibodies directed against TK1 have been described (10-16). The most specific and sensitive TK1 antibodies which have been produced for use in serum TK determinations rely on TK1-peptides representing 15 or 31-amino acids fragments of a certain amino acid sequence in the C-terminal part of TK1 (10, 13). This epitope domain has been named exposed proliferation antigen 210 (XPA210) based on the numbering of the central amino acid in the TK1 peptide sequence. Recently, several studies were published where the level of TK1 in cells and serum were determined by immunological methods using these antibodies and it became clear that the activity and the immunological assays did not correlate but appeared to provide complementing information regarding cell proliferation (15,16 and unpublished results). It was also clear that intact TK1 is poorly immunogenic and the quality of several earlier described antibodies are questionable. The method to produce the TK1 specific antibodies is disclosed in U.S. Pat. No. 6,083,707. The same procedure but using a longer peptide (31 instead of 15 amino acid fragments) has also been disclosed (12). The introduction of methods using XPA210 have led to production of clearly defined immunological reagents that have served as the basis for studies demonstrating that TK1 immunoreactive material can be used to monitor a large number (at least 10 different types) of tumor diseases including breast, lung, bladder, etc. (10-16). Furthermore, based on XPA210 measurements, the recurrence of certain solid tumors (e.g. breast carcinomas) could be predicted several years before other signs of disease could be detected (17). A dot blot immunoassay as well as a histochemistry kit based on this type of purified polyclonal antibodies are available from ssTK Inc (18).
A major problem encountered in many immunological assays is that they may lack sensitivity by masking of epitopes on the antigen in certain situations and/or cross-reactivity of antigen to other proteins occurs.